JP2013525456A - Compositions containing purine and pyrimidine nucleosides, peptides and manganese and their use - Google Patents

Abstract

The present invention is a method for producing a vaccine against a microorganism, comprising culturing, recovering and / or suspending the microorganism in the presence of a radiation protection composition and making the microorganism non-replicatable and / or Methods are provided that include irradiating bacteria or viruses with a dose of radiation sufficient to render them non-infectious. The radioprotective composition used in the method of the invention comprises at least one nucleoside, at least one antioxidant and at least one small peptide. The present invention also provides a method of making a bacterium in culture resistant to ionizing radiation (IR), comprising culturing the bacterium in the presence of a radiation protective composition.
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Description

(Related application)
This application claims priority to US Provisional Patent Application No. 61 / 329,381, filed Apr. 29, 2010, the contents of which are incorporated herein by reference.
(Government support)

  The present invention is in part due to the US Department of Energy Science, Biology and Environmental Improvement Research (BER), grant DE-FG02-04ER63918 from the Environmental Improvement Science Program, and grant FA9559 from the Air Force Science Research Bureau. Made from research funded by -07-1-0128. The government has certain rights in the invention.

  The present invention relates to a method for producing a vaccine against a microorganism, wherein the microorganism is cultured, recovered and / or suspended in the presence of a radiation protective composition, and radiation sufficient to render the microorganism non-replicatable. A method comprising irradiating the microorganism with a dose of The radiation protective composition used in the method of the present invention comprises at least one decapeptide in a mixture of manganese phosphate or manganese bicarbonate buffer. The present invention also provides a method of making a bacterium in culture resistant to ionizing radiation (IR), comprising culturing the bacterium in the presence of a radiation protective composition.

Extremely radiation resistant Deinococcidae can survive acute exposure to ionizing radiation (IR) (10 kGy), ultraviolet (UV) (1 kj / m ² ) and dry (years); and grow under long-term IR (60 Gy / hour) It can be made up of more than 20 different species. In particular, Deinococcus radiodurans is an extremely ionizing radiation (IR) resistant bacterium that can survive exposure to gamma rays that exceed a dose that is cytotoxic and lethal to mammalian cells by a factor of 1000.

  For extremely resistant bacteria such as Deinococcus radiodurans, survival after high doses of IR is due to protection of the protein from oxidation during irradiation, resulting in a much higher recovery than susceptible bacteria With moderate efficiency, the enzyme repair system remains and functions (in this case, cellular proteins are highly sensitive to carbonylation). In a report published in a scientific journal (Daly et al. (2004), Accumulation of Mn (II) in Deinococcusradiodurans facilitates gamma-radiation resistance, Science 306: 925-1084), intracellular manganese (II) is transferred to ionizing radiation. Was involved in fostering radiation resistance by protecting proteins (but not DNA) during the exposure of Drosophila; and a second report published in PLoS Biology (Daly et al. (2007) Protein oxidation implicated As the primary determinant of bacterial radioresistance, PLoS Biology 5 (4) e92), radiation resistance was positively correlated with protein protection during irradiation mediated by non-enzymatic mechanisms.

  Unlike Deinococcus radiodurans, most proteins are not radiation resistant. Similarly, most cells are not radiation resistant, whether eukaryotic, prokaryotic, or mammalian (eg, human). As such, exposure to radiation causes considerable damage to protein structure and / or function. For example, ionizing radiation has been shown to induce (cause) cancer in many different species of animals and in almost every part of the human body.

  In humans, significant overexposure to radiation can result in radiation poisoning (also called “radiation sickness” or “creepingdose”). The term is generally used to refer to acute problems caused by high doses of radiation in the short term, but this also occurs with long term exposure to low levels of radiation. The clinical name for "radiation sickness" is described as acute radiation symptoms according to CDC. Chronic radiation symptoms are present but very rare; this has been observed among employees at the initial radium source production site, as well as in the early stages of the Soviet nuclear program. Short-term exposure can produce acute radiation symptoms; chronic radiation symptoms require long-term high-level exposure.

  Humans routinely encounter radiation in their daily lives, including radiation from electronic devices and mobile phones, as well as natural background radiation. Individuals in close proximity to radioactive elements, such as nuclear facility employees or military personnel, are particularly likely to encounter high doses of radiation. In addition, radiation is used in diagnostic tests such as X-rays and in radiation therapy to treat cancer.

  There are generally very few radioprotective agents suitable for treating humans, and existing ones (eg, amifostine) are cytotoxic and have serious side effects (eg, loss of consciousness, fast or irregular) Breathing, pruritus, nausea and vomiting).

  When radiation exposure is large, there is a significant need for radioprotective agents that are non-toxic, preserve protein function, and are particularly suitable for human use.

  The present invention relates to a method for producing a vaccine against a microorganism, wherein the microorganism is cultured, recovered and / or suspended in the presence of a radiation protective composition, and radiation sufficient to render the microorganism non-replicatable. A method comprising irradiating the microorganism with a dose of The composition for radioprotection used in the vaccine production method of the present invention contains at least one decapeptide in a manganese-containing buffer.

  The present invention also provides a method of making a bacterium in culture resistant to ionizing radiation (IR), comprising culturing the bacterium in the presence of a radiation protective composition. The radiation protective composition used in the IR resistance method of the present invention comprises at least one nucleoside, phosphate, at least one antioxidant and dimethyl sulfoxide (DMSO).

Compounds in Deinococcus radiodurans ultrafiltrate protect proteins, but from ultrafiltrate from Pseudomonas putida (PP), E. coli (EC) and extreme thermophile (Thermus thermophilus) (TT) Indicates that this compound does not protect. Protein-free ultrafiltered Deinococcus radiodurance (DR) cell extract prevents ionizing radiation (IR) -induced protein oxidation in vitro but is a radiation sensitive bacterium Pseudomonas putida (PP), E. coli Extracts from (EC) and advanced thermophile (TT) did not prevent. Purified E. coli protein was incubated in PP-, EC-, TT-, or DR-ultrafiltration extracts during irradiation and subjected to protein carbonyl assay. Coomassie-stained polyacrylamide denaturing gel; carbonyl western blot: demonstrates protein oxidation and protection (no signal). Figure 2 shows the composition of Deinococcus radiodurans (DR) compared to ultrafiltrate from Pseudomonas putida (PP), E. coli (EC) and extreme thermophile (Thermus thermophilus) (TT). E. coli exposed to short-term IR and grown in the presence of various supplements: TGY, standard rich peptide growth medium; DMSO, dimethyl sulfoxide; UMnP, 3 mM uridine / 1 μM Mn ²⁺ / 13 mM PiB (phosphate buffer) The survival curve is shown. Figure 2 shows the role of peptides in resistance to ionizing radiation. (A) Cytosolic distribution and concentration of amino acids in Deinococcus radiodurans: “No IR” Keeped in ice in 25 mM potassium phosphate buffer, pH 7.4, then washed, 25 mM phosphate buffer, pH 7. Non-irradiated control cells kept in 4 (32 ° C.) for 0 or 30 minutes. “+ IR” Irradiated to 7 kGy in ice in 25 mM phosphate buffer, pH 7.4, then washed and held in 25 mM phosphate buffer, pH 7.4 (32 ° C.) for 0 or 30 minutes cell. Cells were harvested, resuspended in 20% TCA and lysed. An aliquot of the neutralized supernatant was analyzed for free amino acid and peptide-derived amino acid content. (B) Radioprotection of BamHI with decapeptide (H-Asp-Glu-His-Gly-Thr-Ala-Val-Met-Leu-Lys-OH; 1261 Da). (C) Glutamine synthase with Mn ²⁺ and leucine (Leu), uridine (U) or decapeptide (DP) in potassium phosphate buffer (PiB), pH 7.4 or sodium bicarbonate (HCO ₃ ), pH 7.4 (GS) radiation protection. Figure 2 shows an approach for the preparation of irradiated vaccines using manganese complexes. (A) Mn ²⁺ complex (Mn-pep-Pi): 3 mM (H-Asp-Glu-His-Gly-Thr-Ala-Val-Met-Leu-Lys-OH) (SEQ ID NO: 1) 1 mM MnCl, 25 mM DNA was prepared from irradiated bacteriophage λ treated (right) or untreated (left) with orthophosphate (Pi) buffer (pH 7.4). DNA (48.5 kbp genome) was purified from bacteriophage λ at the indicated gamma-ray dose (0-40 kGy), subjected to conventional agarose gel electrophoresis, and then Southern blotted with a radiolabeled λ DNA probe. Conclusion: The Mn ²⁺ complex does not significantly protect the DNA packaged in the virus. (B) The same bacteriophage λ preparation as tested in panel A was tested for protein integrity by separating the viral proteins using polyacrylamide gel electrophoresis. Conclusion: Proteins in virus irradiated in the absence of Mn ²⁺ complex (left) were progressively destroyed. In contrast, the protein in the virus sample containing the Mn ²⁺ complex (right) was not affected by the high dose of 40 kGy. (C) At 40,000 Gy, the dose that caused the viral DNA to disappear (panel A) and the virus to be completely non-infectious (not shown), the viral protein remained completely immunogenic. there were. This was tested by Western analysis to challenge the λ protein with antibodies raised in rabbits against non-irradiated λ phage. Note: Identical positive results for immunogenicity were obtained for equivalent Westerns probed with antibodies raised against λ phage exposed to 40,000 Gy in the presence of Mn ²⁺ complexes. In contrast, λ phage exposed to 40,000 Gy in the absence of Mn ²⁺ complex did not produce antibodies in rabbits with significant specificity for native bacteriophage λ. (D) and (E): Transmission electron micrographs (TEM) after irradiation with (E) or untreated (D) λ phage treated with Mn ²⁺ complex. (F) and (G): TEM after (G) or untreated (F) λ phage irradiation (40 kGy) treated with Mn ²⁺ complex. In the presence of the Mn ²⁺ complex, λ phage virus particles exposed to 40 kGy were not damaged. Table and graph data from mice tested with S. aureus (MRSA) are shown. These data indicate that the presence of manganese complexes in the irradiated composition provided a greater immune response in the treated mice.

  The inventors tested the radiation resistance of Deinococcus radiodurans and prepared ultra-purified protein-free cell extracts exhibiting radioprotective properties. Accordingly, the present invention is based, in part, on the discovery of the radioprotective components of Deinococcus radiodurans cell-free extracts as well as artificial compositions containing such components.

  In particular, Applicants have shown that Deinococcus radiodurans ultrapurified and protein-free cell extracts are extremely radioprotective for proteins exposed to gamma radiation. Adenosine, uridine and peptides accumulate in Deinococcus radiodurans ultrafiltrate at higher concentrations than in radiosensitive bacterial ultrafiltrate. In vitro, nucleosides at doses> 10,000 Gy are highly protective for proteins, prevent ionizing radiation (IR) -induced protein carbonylation, and preserve enzyme function in the presence of Mn (II) It was shown that. Radioprotective compositions of adenosine, manganese, peptides and phosphates have been developed. Surprisingly, Deinococcus radiodurans extract has been shown to be a potent radioprotectant for cultured human T cells with greater efficacy than other well-established radioprotective compounds.

The present invention provides synthetic or Deinococcus radiodurans (DR) derived radioprotective compositions and methods of using these compositions to protect proteins and / or cells from radiation damage. These compositions are useful for preventing radiation damage in the compositions as well as in subjects such as humans or in cell culture. The compositions of the present invention contain manganese, at least one antioxidant peptide, or they contain a collection of manganese and individual amino acids. In additional embodiments, the composition may also include at least one nucleoside. As used herein, the term “radioprotective composition” or “radiationprotective composition” refers to the DR limit prepared according to the methods described herein. It can mean a filtered extract or it can mean a synthetic composition comprising a collection of manganese and at least one antioxidant peptide or individual amino acids. If DR ultrafiltration extract is used, this extract can be supplemented with any of the compounds described and disclosed herein. For example, DR ultrafiltrate can be prepared according to the methods disclosed herein, for example, additional Mn ²⁺ or peptides can be added to the extract.

  The radiation protection composition further contains leucine, alanine and / or valine. Leucine is strongly involved in scavenging hydrogen peroxide in the presence of Mn (II) and may be a component of larger intracellular complexes including uridine and adenosine. Strong in vitro evidence indicates a synergy between adenosine and manganese and phosphate. The stoichiometry of adenosine and manganese and phosphate or bicarbonate buffer can be optimized for apoptosis assays.

  Applicants have shown that adenosine alone and Mn (II) alone are radioprotective in vivo for mammalian cell lines and for bacterial cell culture.

  Without being bound by a particular theory, compositions comprising purine nucleosides (eg adenosine), pyrimidine nucleosides (eg uridine) and peptide antioxidants (eg manganese-peptides) can conceal the active site and surface of proteins. Acts as a radiation protection agent. Purine nucleosides such as adenosine (and optionally in combination with pyrimidine nucleosides uridine and peptides) mediate their radioprotective action upon accumulation in the cell, which suppresses radiation-induced protein oxidation, and Preserve enzyme function in the presence of Mn (II). Adenosine seems to protect the protein and thus scavenge a small population of ROS.

  Furthermore, without being bound by any particular theory, under aerobic or anaerobic irradiation conditions, superoxide is produced in the cell during irradiation, which does not easily pass through the membrane. Because. Superoxide does not react with DNA, but superoxide damages and inactivates enzymes with exposed 2Fe-2S or 4Fe-4S clusters to release Fe (II), such as, but not limited to, cysteine Certain exposed amino acids such as can also be damaged. The problem with iron in the cell is that if it is not bound and is “free”, it causes a Fenton reaction in the presence of hydrogen peroxide to generate hydroxyl radicals. Thus, the state of liberating bound Fe (II) is extremely dangerous because the loss of Fe from Fe-dependent enzymes, as well as the generation of hydroxyl radicals, leads to failure of the biochemical pathway they manipulate. It is. The method of the application optimally protects against these dangerous conditions.

  Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described.

  As used herein, “a” or “an” means at least one unless explicitly stated otherwise. The term “about” refers to a value that is 10% or less greater or less than the value modified by the term, unless otherwise specified. For example, the term “about 5% (w / w)” means a range of 4.5% (w / w) to 5.5% (w / w).

  The present invention provides a method for preserving protein function or protein immunogenicity, comprising contacting a protein with a composition of the present invention. One embodiment of the invention is a method of preserving protein function when exposed to extreme conditions such as gamma radiation. In another embodiment of the invention, the method preserves protein function during drying.

  A method of preserving protein function provides radiation protection when the protein is exposed to high doses of radiation, for example, doses greater than 10 kGy, for example 17.5 kGy.

  In another embodiment, the present invention provides a method for protecting protein function or protein immunogenicity in a cell culture or virus preparation, comprising any of the radioprotective compositions described herein. Use to provide a method that includes culturing, harvesting, and / or suspending cells. Viral preparations can relate to single-stranded or double-stranded DNA or RNA genomes. The cell culture can be prokaryotic or eukaryotic. In one embodiment, the cell culture is bacterial. In another embodiment, the cell culture is mammalian. In yet another embodiment, the cell culture is a culture for the purpose of propagating viruses.

  Any nucleoside, if present, can be used in the radiation protection composition. Suitable nucleosides include, but are not limited to, adenosine, uridine, β-pseudouridine, inosine, and mixtures thereof. In addition, nucleosides containing two carbonyl oxygen groups (C═O) separated by one (N 3) H group can also be used. In one embodiment, the nucleoside is adenosine or uridine. In one embodiment, the composition contains adenosine. In another embodiment of the invention, the composition contains uridine. The amount of nucleoside in the composition will vary with respect to its use. One skilled in the art can determine the appropriate amount. In some embodiments of the invention, the amount of nucleoside ranges from about 0.01 mM to about 15 mM, from about 0.1 mM to about 1 mM, from about 1 mM to about 10 mM, from about 1 mM to about 15 mM. In one embodiment, the concentration of one or more nucleosides comprises from about 1 mM to about 15 mM adenosine and / or uridine.

A variety of antioxidants can be used or present in the composition. Suitable antioxidants include manganese, vitamin E and manganese phosphate, Mn-peptide, Mn-amino acid (eg leucine), Mn-TRIS, Mn-melanin, Mn-caffeine, Mn-ribose, Mn-trehalose, Mn-dipicolinic acid, Mn-phosphate and Mn-bicarbonate are mentioned. In one embodiment of the invention, the antioxidant is manganese. In another embodiment, the antioxidant is MnCl _2. In yet another embodiment, the antioxidant is vitamin E and / or aspirin. The amount of antioxidant in the composition will vary upon its use. One skilled in the art can determine the appropriate amount. In one embodiment, the composition contains about 0.01 mM to about 15 mM antioxidant. In another embodiment, the composition contains from about 0.01 mM to about 12.5 mM.

  In one embodiment of the invention, the antioxidant is manganese phosphate that can be provided as a mixture. In one embodiment, the mixture is produced by mixing a solution of manganese and a solution of phosphate. The amount of antioxidant in the composition will vary with respect to its use. One skilled in the art can determine the appropriate amount. In one embodiment, the composition comprises about 0.01 mM to about 15 mM manganese (Mn (II)) ions. In a more specific embodiment, the composition comprises about 0.01 mM to about 15 mM manganese (Mn (II)) ions in phosphate buffer. In a more specific embodiment, the composition comprises phosphate buffer at a concentration of about 1 mM to about 25 mM. In one specific embodiment, the mixture is a 1 mM solution of Mn (II) and a solution of 25 mM phosphate buffer (pH 7.4).

  The composition contains one or more amino acids that exhibit cytoprotective properties. In one embodiment of the invention, the composition is further selected from the group consisting of asparagine, glutamine, serine, histidine, glycine, threonine, arginine, tyrosine, methionine, phenylalanine, isoleucine, lysine, ornithine, leucine, valine and alanine. At least one or more amino acids. In another embodiment, the amino acid is leucine. In an alternative embodiment, the amino acid is glycine. In another embodiment, the composition comprises at least leucine and alanine. In another embodiment, the composition does not contain proline. In yet another embodiment, the composition contains no more than 10% proline as measured against the presence of another amino acid. For example, an equal mixture of 12 different amino acids contains no more than one proline residue in this embodiment.

  As an alternative to, or in addition to, the presence of individual amino acids, the compositions and methods using these compositions can include at least one small peptide, such as, but not limited to, a decapeptide. As used herein, “small peptide” means a small linear amino acid with a residue length of about 25 or less. In one embodiment, the small peptide used in the composition or method of the invention is about 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, It is 10, 9, 8, 7, 6, 5, 4, 3 or 2 amino acids long. The actual sequence of the peptide is not critical to the compositions and methods of the present invention, so any random peptide chain is sufficient. For example, in one embodiment, the compositions and methods using these compositions can include at least one small peptide, wherein the small peptide is SEQ ID NO: 1: Asp-Glu-His-Gly-Thr- It comprises an amino acid sequence that is at least about 80% identical to the amino acid sequence of Ala-Val-Met-Leu-Lys (SEQ ID NO: 1). In one embodiment, the small peptide does not contain a proline residue. In another embodiment, the peptide contains less than 10% proline compared to other amino acids. For example, in this specific embodiment, the 12-mer contains no more than one proline residue.

  In a further embodiment, each small peptide is independently at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 and the amino acid sequence of SEQ ID NO: 1. Amino acid sequences that are%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical. Small peptides that are less than 100% identical to the amino acid sequence of SEQ ID NO: 1 are considered to be variants thereof.

  The amount of small peptide varies. An appropriate amount can be determined by one skilled in the art depending on various factors such as the subject, duration of radiation exposure, amount of radiation exposure, and the like. In some embodiments of the invention, the amount of small peptide ranges from about 0.01 mM to about 15 mM, from about 0.1 mM to about 1 mM, from about 1 mM to about 10 mM, from about 1 mM to about 15 mM. In one embodiment, the concentration of one or more small peptides comprises from about 1 mM to about 15 mM of the peptide of SEQ ID NO: 1 or variants thereof. In other embodiments, the concentration of the one or more small peptides is about 15 mM or less, about 14 mM or less, about 13 mM or less, about 12 mM or less, about 11 mM or less, about 10 mM or less, about 9 mM or less, about 8 mM or less, about 7 mM. Hereinafter, the peptide of SEQ ID NO: 1 of about 6 mM or less, about 5 mM or less, about 4 mM or less, about 3 mM or less, about 2 mM or less, about 1 mM or less, about 0.5 mM or less is included. Of course, the concentration of the one or more small peptides can be between any of the listed concentrations, such as from about 15 mM to about 14 mM, from about 14 mM to about 13 mM, from about 13 mM to about 12 mM, from about 12 mM to about 11 mM, from about 11 mM to about 11 mM. About 10 mM, about 10 mM to about 9 mM, about 9 mM to about 8 mM, about 8 mM to about 7 mM, about 7 mM to about 6 mM, about 6 mM to about 5 mM, about 5 mM to about 4 mM, about 4 mM to about 3 mM, about 3 mM to about 2 mM , About 2 mM to about 1 mM, about 1 mM to about 0.5 mM, or the like, or a variant thereof.

  A polypeptide having an amino acid sequence that is at least, for example, about 95% “identical” to a reference amino acid sequence, eg, SEQ ID NO: 1, is identical in amino acid sequence to the reference sequence, provided that the amino acid sequence is referenced It is understood to mean that it comprises up to about 5 modifications for each 100 amino acids of the amino acid sequence. In other words, to obtain a peptide having an amino acid sequence that is at least about 90% identical to the reference amino acid sequence, up to about 10% of the amino acid residues of the reference sequence can be deleted or replaced with another amino acid. Alternatively, multiple amino acids up to about 10% of the total amino acids in the reference sequence can be inserted into the reference sequence. These modifications of the reference sequence occur at the N-terminal or C-terminal position of the reference amino acid sequence, or anywhere between the terminal positions, independently between amino acids in the reference sequence, or within the reference sequence. Can be plugged in a continuous group.

  As used herein, “identity” is a measure of the identity of a nucleotide or amino acid sequence as compared to a reference nucleotide or amino acid sequence. In general, the sequences are aligned so that the highest order match is obtained. “Identity” per se has a recognized breath in the art and can be calculated using published techniques (eg, Computational Molecular Biology, Lesk, AM, ed., Oxford University Press, New York ( 1988); Biocomputing: Informatics AndGenome Projects, Smith, DW, ed., Academic Press, New York (1993); Computer Analysis of Sequence Data, Part I, Griffin, AM, and Griffin, HG, eds., Humana Press, New Jersey (1994); von Heinje, G., Sequence Analysis In Molecular Biology, Academic Press (1987); and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., MStockton Press, New York (1991)). There are several ways to measure identity between two polynucleotide or polypeptide sequences, but the term “identity” is well known to those skilled in the art (Carillo, H. & Lipton, D., Siam J Applied Math 48: 1073 (1988)). Commonly used methods for determining identity or similarity between two sequences include Guide to Huge Computers, Martin J. Bishop, ed., Academic Press, San Diego (1994) and Carillo, H. & Lipton, Examples include, but are not limited to, methods disclosed in D., Siam JApplied Math 48: 1073 (1988). Computer programs may also contain methods and algorithms for calculating identity and similarity. Examples of computer program methods for determining identity and similarity between two sequences include the GCG program package (Devereux, J., et al., Nucleic Acids Research 12 (i): 387 (1984)), Examples include, but are not limited to, BLASTP, ExPASy, BLASTN, FASTA (Atschul, SF, et al., J Molec Biol 215: 403 (1990)) and FASTDB. Examples of methods for determining identity and similarity are Michaels, G. and Garian, R., Current Protocols in Protein Science, Vol 1, John Wiley & Sons, Inc. (2000). (Incorporated herein by reference).

  In one embodiment of the invention, the algorithm used to determine identity between two or more polypeptides is BLASTP. In another embodiment of the invention, the algorithm used to determine identity between two or more polypeptides is FASTDB, which is described in Brutlag et al. (Comp. App. Biosci. 6: 237). -245 (1990)), the content of which is incorporated herein by reference. In the FASTDB sequence alignment, the query and reference sequences are amino sequences. Sequence alignment results are given in% identity. Parameters that can be used for FASTDB alignment of amino acid sequences to calculate% identity include, but are not limited to: matrix = PAM, k-pair = 2, mismatch penalty = 1, linkage Penalty = 20, randomized group length = 0, cut-off score = 1, gap penalty = 5, gap size penalty = 0.05, window size = 500, or subject amino sequence length (whichever is shorter) .

  For N-terminal or C-terminal additions or deletions (but not for internal additions or deletions), manual correction can be made if the reference sequence is shorter or longer than the query sequence, but this This is because the FASTDB program does not account for N-terminal and C-terminal truncation or addition of the reference sequence when calculating%. For query sequences that are truncated at the N- or C-terminus relative to the reference sequence, calculate the number of residues in the query sequence at the N- and C-terminus relative to the reference sequence that is not aligned / aligned. This corrects the% identity as% of the total base of the query sequence. The result of FASTDB sequence alignment establishes alignment / alignment. The alignment percentage is then subtracted from the percent identity calculated by the FASTDB program using specific parameters to arrive at the final percent identity score. This corrected score can be used for the purpose of determining how the alignments "correspond" to each other as well as the identity percentage. Reference sequence residues extending beyond the N- or C-terminus of the query sequence can be considered for the purpose of manually adjusting the% identity score. That is, residues that are not aligned / aligned with the N- or C-terminus of the comparison sequence can be counted when manually adjusting the% identity score or alignment numbering.

  For example, a 90 amino acid residue query sequence is aligned with a 100 residue reference sequence to determine percent identity. Deletions are commercial at the N-terminus of the query sequence, and therefore the FASTDB alignment does not show the first 10 residue alignment / alignment at the N-terminus. Ten unpaired residues represent 10% of the reference sequence (number of residues at unmatched N- and C-terminals / total number of residues in the reference sequence), where 10% is calculated by the FASTDB program Is deducted from the percent identity score. If the remaining 90 residues are perfectly matched (100% alignment), the final% identity is 90% (100% alignment—10% mismatched overhang). In another example, a 90 residue query sequence is compared to a 100 reference sequence, provided that the deletion is an internal deletion. In this case, the percent identity calculated by FASTDB is not manually corrected because there are no residues at the N- or C-termini of subject sequences that are not aligned / aligned with the query. In yet another example, a 110 amino acid query sequence is aligned with a 100 residue reference sequence to determine percent identity. Additions in the query occur at the N-terminus of the query sequence, so the FASTDB alignment cannot show the first 10 residue alignment / alignment at the N-terminus. If the remaining 100 amino acid residues of the query sequence have 95% identity to the full length of the reference sequence, the N-terminal addition of the query is ignored and the percent identity of the query to the reference sequence is 95%.

In one embodiment, the composition comprises adenosine, uridine, leucine, adenine and manganese. In another embodiment, the composition comprises from about 1 to about 15mM adenosine and about 1 to about 12.5 mM MnCl _2. In another embodiment, the composition comprises a Deinococcus radiodurans extract containing one or more nucleosides and one or more antioxidants.

  By using the method of the present invention, any protein function can be preserved. In a preferred embodiment of the invention, the protein is an enzyme. The disclosed methods of the present invention are particularly useful in preventing protein oxidation associated with ultraviolet light and aging. In addition, the method also preserves protein function during drying, thus helping to increase the shelf life of dried blood products and enzyme-based drugs (which are stored dry).

  The methods of the present invention optimally preserve protein function (eg, enzyme activity) during exposure to radiation. One embodiment of the present invention is a storage method comprising contacting a protein (eg, an enzyme) with a composition comprising one or more nucleosides and one or more antioxidants.

  Another embodiment of the present invention is a method for increasing the durability and lifetime of microbial and enzyme-derived fuel cells, wherein the fuel cell components comprise one or more nucleosides and one or more antioxidants. A method comprising contacting with a composition.

  This method is suitable for preserving the function of a large number of proteins, such as, but not limited to, Fe-S complexes (metabolic enzymes), as well as enzymatic repair functions determined by redox-active (4Fe-4S) clusters. obtain. Exemplary proteins include proteins associated with the production of reactive oxygen species (ROS), transport protein precursors that can reduce biosynthetic requirements and suppress ROS production, proteins that protect against ROS, damaged molecules ( Non-DNA) repair and redox regulation and proteins involved in Mn and Fe dependent systems. Other exemplary proteins are listed in Ghosal et al. (2005), FEMS Microbiology Reviews 29: 361-375, the description of which is incorporated herein by reference.

  The present invention is a method for producing a vaccine against a microorganism, comprising culturing, recovering and / or suspending the microorganism in the presence of the radiation protection composition of the present invention and making the microorganism non-replicatable. Also provided is a method comprising irradiating a microorganism with a sufficient dose of radiation. In one embodiment, the radiation protection composition is synthetic. In another embodiment, the radiation protection composition is a DR ultrafiltered extract.

  Methods for preparing vaccines are well known in the art. The methods provided herein can be applied to these well-known vaccine preparation methods, or they can be used separately and independently of traditional vaccine preparation methods. For example, one embodiment of the present invention provides a vaccine preparation method that does not genetically engineer the microorganisms for which the vaccine is to be prepared. The methods disclosed herein are normal in the presence of a radiation protective composition so that the three-dimensional structure of proteins in microorganisms as well as the cell surface markers on microorganisms are preserved in extreme doses of radiation. Wild-type microorganisms are cultured, recovered and / or suspended. The radiation dose is intended to destroy the genome of the microorganism so that the microorganism cannot replicate. Following radiation dose administration, non-replicaable cells can be collected and vaccine preparation can be performed using normal vaccine preparation techniques. The protective composition of the present invention preserves at least a fragment of a microbial immunogenic protein so that administration of a vaccine containing irradiated microorganisms to an animal produces an immunogenic response. Thus, the vaccine preparation method of the present invention can be practiced using conventional cell culture techniques. Microorganisms for which vaccines can be prepared using the methods of the present invention include bacteria and viruses. Standard cell culture techniques for bacteria and viruses are well known in the art.

  Of course, the vaccine preparation method of the present invention is not limited to a particular type of radiation, although the type and dose used can render the microorganism non-replicatable. Examples of radiation include, but are not limited to, ultraviolet rays, alpha rays, beta rays, gamma rays, X rays and neutron rays. In one embodiment, the radiation dose is at least about 20 kGy. The dose of radiation can exceed 25,000 Gy (25 kGy) for the bacterial mixture and the dose of radiation for the viral mixture can exceed 40,000 Gy (40 kGy).

  The present invention also provides a method of making a bacterium in culture resistant to ionizing radiation (IR), comprising culturing the bacterium in the presence of the radiation protective composition of the present invention. The radiation protective composition used in the IR resistance method of the present invention comprises at least one nucleoside, phosphate, at least one antioxidant and a non-metabolized hydroxyl radical scavenger such as dimethyl sulfoxide (DMSO) Including, but not limited to).

  The present invention also provides a method of treating or preventing the effects of radiation exposure. The method includes treating or preventing the effects of radiation exposure with a therapeutic agent comprising one or more nucleosides and one or more antioxidants.

  In one embodiment of the invention, the radiation exposure is by UV exposure. In another embodiment of the invention, the radiation exposure is by ionizing radiation. In another embodiment of the invention, the radiation exposure is long term.

  As used herein, the term “therapeutic agent” refers to a composition comprising one or more nucleosides and one or more antioxidants, as well as other pharmaceutically acceptable components such as pharmaceutically acceptable. Formulations containing various carriers.

  As used herein, the term “radiation exposure” means exposure to any radiation at a dose and duration sufficient to cause damage. Radiation exposure includes, but is not limited to, exposure to ultraviolet radiation, alpha radiation, beta radiation, gamma radiation, X-rays and neutron radiation.

  In one embodiment, the present invention provides a method of treating or preventing the side effects of radiation therapy. As used herein, the term “radiotherapy” refers to the use of some form of energy (eg, ionizing radiation) to kill cancer cells and shrink tumors. The term “radiotherapy” refers to external radiation therapy (eg, intraoperative radiation therapy and prophylactic cranial radiation (PC)), internal radiation therapy (interstitial radiation therapy, intraluminal radiation therapy or intraluminal radiation therapy), whole body radiation. Includes all types of radiation therapy, including but not limited to treatment, stereotactic (or stereotaxic) radiosurgery, three-dimensional (3D) conformal radiation therapy, intensity modulated radiation therapy (IMRT). Furthermore, the term “radiotherapy” also encompasses radiotherapy using various radiation sources such as, but not limited to, X-rays, gamma rays, particle beams, proton beam treatments and high energy photon rays. Radiation therapy is used to treat various cancers, such as solid tumors (eg, brain, breast, cervix, larynx, lung, pancreas, prostate, skin, spine, stomach, uterine cancer, or soft tissue sarcoma). Radiation therapy is also used to treat leukemias and lymphomas (ie, blood-forming cells and lymphoid cancers, respectively), as well as skin, cervical, and thyroid cancers.

  As used herein, the term “radiotherapy side effects” refers to any side effects experienced by a subject undergoing radiotherapy. Such side effects include, but are not limited to, fatigue and skin reactions, anemia, increased risk of injury or bleeding, reduced fertility, dry mouth, appetite and weight loss, hair loss and the like.

  A “subject in need of treatment” is an animal that has a bacterial infection that is potentially life threatening or impairs the health of the animal or shortens its lifespan. The animal can be a fish, bird or mammal. Exemplary mammals include humans, domesticated animals (eg, cows, horses, sheep, pigs, dogs and cats), and exhibit animals at, for example, zoos. In a preferred embodiment, the subject is a human.

  The terms “treating”, “treatment” and “therapy” as used herein refer to curative therapy, prophylactic therapy and preventative therapy.

  As used herein, unless otherwise indicated, the term composition refers to, but is not limited to, pharmaceutical and functional food compositions containing one or more nucleosides and one or more antioxidants. Is intended to encompass. A composition is an “inactive ingredient” or lacking pharmacological activity or other direct action in diagnosing, curing, alleviating, treating or preventing a disease, or to affect the structure or any function of the human body One or more “excipients” that are “compounds” may also be included.

  “Pharmaceutically acceptable” components are suitable for use with humans, animals and / or plants without undue side effects (eg, toxicity, irritation and allergic reactions) corresponding to a reasonable benefit / risk ratio. There is something.

  The therapeutic agent can contain any nucleoside. Suitable nucleosides include, but are not limited to, adenosine, uridine, β-pseudouridine, inosine, and mixtures thereof. In one embodiment, the nucleoside is adenosine and / or uridine. In one embodiment, the therapeutic agent contains adenosine. In other embodiments of the invention, the therapeutic agent contains uridine.

The therapeutic agent can contain a variety of suitable antioxidants disclosed herein. For example, suitable antioxidants include manganese, vitamin E and manganese phosphate, Mn-peptide, Mn-amino acid (eg leucine), Mn-TRIS, Mn-melanin, Mn-caffeine, Mn-ribose, Mn-trehalose. , Mn-dipicolinic acid, Mn-phosphate and Mn-bicarbonate. In one embodiment of the invention, the therapeutic antioxidant is manganese. In another embodiment, the antioxidant is MnCl _2. In yet another embodiment, the antioxidant is one or more peptides.

In one embodiment of the present invention, an important antioxidant is manganese phosphate that can be provided in near millimolar concentrations. In another embodiment, the antioxidant is MnCl ₂ and the phosphate is added separately. The phosphate may or may not be an orthophosphate. The amount of antioxidant in the composition will vary upon its use. One skilled in the art can determine the appropriate amount. In one embodiment, the composition contains about 0.01 mM to about 15 mM manganese (Mn (II)) ions and about 1 mM to about 25 mM phosphate buffer.

  The amount of nucleoside and antioxidant in the therapeutic agent varies. Depending on the subject, duration of radiation exposure, amount of radiation exposure, etc., one skilled in the art can determine an appropriate amount. In some embodiments of the invention, the amount of nucleoside ranges from about 0.01 mM to about 15 mM, from about 0.1 mM to about 1 mM, from about 1 mM to about 10 mM, from about 1 mM to about 15 mM. In one embodiment, the concentration of one or more nucleosides comprises from about 1 mM to about 15 mM adenosine and / or uridine. In another embodiment, the concentration of antioxidant ranges from about 0.01 mM to about 15 mM. In another embodiment, the therapeutic agent contains about 0.01 mM to about 12.5 mM.

  The therapeutic agent may further contain one or more amino acids that exhibit cytoprotective properties. In one embodiment of the invention, the therapeutic agent further contains at least one or more amino acids selected from the group consisting of leucine, valine and alanine. In another embodiment, the amino acid is leucine. In another embodiment, the amino acid is glycine.

In one embodiment, the therapeutic agent comprises adenosine, uridine, leucine, adenine and manganese. In alternative embodiments, the therapeutic agent comprises about 1mM~ about 15mM adenosine and about 1mM~ about 12.5 mM MnCl _2. In another embodiment, the composition comprises a Deinococcus radiodurans extract containing one or more nucleosides and one or more antioxidants.

  In yet another embodiment of the invention, the therapeutic agent comprises one or more nucleosides (eg, adenosine, uridine, β-pseudouridine, inosine and mixtures thereof), one or more antioxidants (eg, manganese, A composition suitable for human use comprising peptide and vitamin E) and optionally one or more amino acids selected from the group consisting of leucine, valine and alanine. In one embodiment, a composition suitable for human use comprises adenosine and manganese.

  In an alternative embodiment of the present invention, the therapeutic agent is a Deinococcus radiodurans extract containing one or more nucleosides and one or more antioxidants.

  Methods for treating or preventing the effects of radiation exposure include administration of a therapeutic agent comprising one or more nucleosides and one or more antioxidants to a subject in need thereof.

  One embodiment is a method of preventing side effects of radiation therapy, wherein a Denococcus radiodurance extract comprising one or more nucleosides and one or more antioxidants is administered to a subject in need thereof. It is a method including.

Another embodiment of the present invention is a method for preventing side effects of radiation therapy to one or more nucleosides, antioxidants and optionally alanine, valine and leucine to a subject in need thereof. A method comprising administration of a composition comprising an amino acid selected from the group consisting of: Preferably, the one or more nucleosides are adenosine and / or uridine, which may be present in an amount of about 1 mM to about 15 mM adenosine and / or uridine. The one or more nucleosides may also be selected from the group consisting of adenosine, uridine, β-pseudouridine, inosine, and mixtures thereof. The antioxidant can be manganese (eg, about 1 mM to about 12.5 mM). In one embodiment, the antioxidant is MnCl _2. In another embodiment, the antioxidant is one or more peptides. In another embodiment, the composition comprises adenosine, uridine, leucine, adenine and manganese.

  The method of the application is particularly beneficial. Compared to well-established radioprotectors (eg, amifostine), compositions comprising one or more nucleosides and one or more antioxidants (eg, adenosine, uridine, peptides and Mn) are relatively non- Toxic.

  The method of the present invention is particularly suitable for pre- and post-exposure treatment of military employees and civilians exposed to ionizing radiation accidentally or deliberately.

  The method can also be used prophylactically for individuals exposed to significant chronic levels of radiation, for example in nuclear facilities, during long-term space flights, or at the International Space Station.

  A “safe and effective amount”, when used in the methods of the present invention, produces the desired therapeutic response without undue side effects (eg, toxic, irritant or allergic reactions) equivalent to a reasonable benefit / risk ratio. The amount of a component that is sufficient for A “therapeutically effective amount” is an amount of a component that is effective to produce a desired therapeutic response, eg, to slow down the rate of bacterial cell division, to cause bacterial cell division to stop, or to Means an amount effective to cause death or reduce the rate of population growth. A specific safe and effective amount or therapeutically effective amount depends on the particular condition being treated, the physical condition of the subject, the type of subject being treated, the duration of the treatment, the nature of the comorbid treatment (if any), and the use It varies with factors such as the specific formulation and the structure of the compound or its derivatives.

  Application means include, but are not limited to, direct, indirect, carrier and special means or any combination of means. Direct application of the phage is by nasal spray, nasal drop, nasal ointment, nasal wash, nasal injection, nasal tampon, bronchial spray or inhaler, or by use of pharyngeal lozenges, or by use of mouth washes or gargles, or It can be done by the use of an ointment applied to the nostril, nasal bridge, or face, or indirectly by any combination thereof, as well as by similar application methods. The forms in which the phage is administered include, but are not limited to, lozenges, troches, candy, injections, chewing gums, tablets, powders, sprays, liquids, ointments and aerosols.

  The therapeutic agent can also be placed in a nasal spray, in which case the nasal spray is a carrier. Nasal sprays can be long acting or timed release sprays and can be made by means well known in the art. Since inhalants can also be used, the therapeutic agent can also reach the bronchi, including the lungs.

  The therapeutic agent can be added to these materials in liquid form or in a lyophilized state, where it is solubilized when it encounters bodily fluids such as saliva. Enzymes can also be present in micelles or liposomes.

  Although these methods can be used on any mammalian species, such as farm animals such as horses, sheep, pigs, chickens and cows, the preferred use of the composition is for humans.

  The effective dosage rate and amount of the composition will depend, in part, on whether the composition is used therapeutically, prophylactically, the duration of exposure of the recipient to radiation, the type of radiation, the size and weight of the individual, etc. Determined. The duration of use of the composition can also be either prophylactic (in this case, use can be hourly, daily or weekly, for a short time), or the use can be therapeutic (in this case, usefulness) A stronger regimen of use of the composition is needed so that can last for hours, days or weeks and / or on a daily basis or for a timed period between days). Any dosage form used should provide the minimum number of units during the minimum amount of time. The concentration of active units of phage that would provide an effective or dose of phage ranges from about 100 units / ml fluid to about 100,000 units / ml in a wet or humid environment of the nasal and oral passages, possibly It can range from about 100 units / ml to about 10,000 units / ml. More specifically, time exposure to radiation can affect the desired concentration of active radioprotective composition units / ml. Carriers classified as “long-term” or “delayed” release carriers (eg, certain nasal sprays or lozenges) retain or provide a low concentration of the composition, but over a longer period of time, On the other hand, it should be noted that “short term” or “rapid” release carriers (eg, gargles) retain or provide a high concentration of composition / ml, but for a short time. It is further understood that a therapeutically effective amount of a particular composition can be determined by one of ordinary skill in the art after careful consideration of factors associated with the subject.

  The selection of a preferred effective dose can be determined by one skilled in the art (eg, through clinical trials) based on consideration of several factors known to those skilled in the art. Such factors include those that are known to those skilled in the art to reflect the disease to be treated or prevented, related symptoms, patient weight, patient immune status, and the accuracy of the pharmaceutical composition being administered. Factors are mentioned.

  The exact dose to be used in the formulation will also depend on the route of administration and should be determined by the judgment of the practitioner and the environment of each patient. Effective doses can be extrapolated from dose-response curves derived from in vitro or animal model test systems.

  For prophylactic and therapeutic treatment and / or prevention of the effects of radiation exposure, compositions comprising nucleosides and antioxidants can be applied directly, indirectly, by carriers and special means or any combination of species. Can be done. Direct application of phage can be done by using nasal sprays, nasal drops, nasal ointments, nasal rinses, nasal injections, nasal tampons, bronchial sprays or inhalers, or the use of pharyngeal lozenges or of mouthwash or gargle It can be done by use or by the use of an ointment applied to the nostril, nasal bridge or face, or indirectly by any combination thereof, as well as by similar application methods. The forms in which the phage is administered include, but are not limited to, lozenges, troches, candy, injections, chewing gums, tablets, powders, sprays, liquids, ointments and aerosols. For therapeutic treatment of anthrax, bronchial sprays and aerosols are most beneficial because the means for dispensing these carriers or compositions allows the phage to reach the bronchi and lungs.

  The compositions of the present invention can be administered by parenteral, subcutaneous, intravenous, intramuscular, intraperitoneal, transdermal or buccal routes. For example, the agent can be administered locally to the site of injury by microinjection. Alternatively, or coexisting, administration is by the oral route. The dose administered will depend on the age, health and weight of the recipient, the type of co-treatment (if any), the frequency of treatment, and the nature of the desired effect.

  In one embodiment of the invention, the method includes administration of a therapeutic agent in a pharmaceutically acceptable carrier. Suitable carriers and their formulations are described in Remington's Pharmaceutical Sciences, 2005, Mack Publishing Co. Typically, an appropriate amount of a pharmaceutically acceptable salt is used in the formulation to make the formulation isotonic. Examples of pharmaceutically acceptable carriers include liquids such as saline, Ringer's solution and dextrose solution. The pH of the solution is preferably from about 5 to about 8, more preferably from about 7 to about 7.5. The formulation can also include a lyophilized powder. Further carriers include sustained release preparations, such as semi-permeable matrices of solid hydrophobic polymers, which are in the form of shaped articles, such as films, liposomes or microparticles. It will be apparent to those skilled in the art that certain carriers can be more heavily selected depending upon, for example, the route of administration as well as the concentration of the proinflammatory cytokine inhibitor being administered.

  The method is optimal to provide treatment for numerous redox-related cell damage mediated by protein injury and to promote wound healing.

  One embodiment of the present invention is a method for preparing a Deinococcus radiodurans cell free ultrafiltered extract exhibiting radioprotective properties. In one embodiment, the method recovers the Deinococcus radiodurance by centrifugation, for example, lyses the Deinococcus radiodurance culture to form a lysate, and after washing the Deinococcus radiodurance lysate, produces a supernatant. Centrifuging the lysate for a time and under conditions sufficient. After centrifugation, the supernatant is passed through a microfilter, preferably a 3 kilodalton microfilter, and boiled for an appropriate time. In one embodiment, the supernatant is boiled for about 15 to about 45 minutes after filtration. The resulting Deinococcus radiodurans extract contains one or more nucleosides and one or more antioxidants, dissolves in butanol, resists boiling and is cell free.

  In one embodiment, the extract contains adenosine and manganese. In another embodiment, the extract contains adenosine and / or uridine manganese. The cell extract may further contain leucine, alanine and / or valine. In one embodiment, the Deinococcus radiodurans extract contains at least adenosine, uridine, leucine, adenine and manganese.

  Without further explanation, using the foregoing description as well as the following illustrative examples, one of ordinary skill in the art will be able to make and use the invention and perform the claimed methods. Accordingly, the following working examples illustrate preferred embodiments of the present invention and do not limit the remaining disclosure in any way.

Example 1-Preparation of Protein-free Extract from Deinococcus radiodurance Deinococcus radiodurance (ATTC BAA-816) was grown in TGY to OD600 0.9, recovered by centrifugation and French pressurization Dissolved by processing. Cells were washed and then lysed in _double distilled deionized sterile water (dH ₂ O). Prior to lysis, the cell water content was adjusted with dH ₂ O to yield a lysate showing an intracellular concentration of about 50%. The crude cell extract was centrifuged at 175,000 × g for 20 hours. The supernatant was passed through a <3 kilodalton Microcon centrifuge filter (Millipore, USA) and boiled for 30 minutes. The Coomassie (Bradford) protein assay was used to confirm the virtual absence of protein in ultrapurified extracts that were aliquoted and stored at minus 80 ° C.

Example 2-Analysis of protein-free extracts from Deinococcus radiodurans Ultrafiltered cell extracts were obtained from Deinococcus radiodurans (ATCC BAA-816), Pseudomonas putida (ATCC 47054), E. coli (MG 1655) and It was prepared from highly thermophilic bacteria (Thermus thermophilus) (ATCC BAA-163). ME Maguire provided wild type E. coli (MM 1925, strain K12) and its isogenic mntH mutant (MM2115). Deinococcus radiodurans recA- (rec30) and E. coli recA- (DH10B) are known in the art. The Jurkat T cell line was ATCC TIB-152. DR-, PP-, EC- and TT-ultrafiltrate were prepared from bacteria grown as a batch culture in TGY medium at the same optical density (0.9; log phase) at 600 nm. Due to the large scale production of DR-ultrafiltrate used for E. coli and Jurkat T cell radioprotection studies, the high cell density growth of Deinococcus radiodurans was in a 20 L fermentor. Cells were broken apart by passing through a French press. Bacterial lysates are centrifuged at 12,000 × g (1 hour at 4 ° C.) in the following order; supernatants are normalized for protein-based concentrations to 190,000 × g (4 ° C. For 48 hours); and the ultracentrifuge supernatant was filtered through a 3 kDa filter. The ultrafiltrate was boiled for 40 minutes, concentrated 5 times and stored at -80 ° C. The chemical composition of DR-, PP-, EC- and TT-ultrafiltrate was determined as follows: Mn and Fe in Perkin Elmer model 4100ZL atomic absorption photometer; inorganic phosphate by Malachite Green assay; HPLC Bases, nucleosides and nucleotides by: protease activity with azocasein as substrate; and amino acids by pre-column derivatization as performed by Agilent Technologies.

Example 3-Radioprotective action in E. coli Independently and in combination, the radioprotective properties of Mn ²⁺ , phosphate, uridine and DMSO were determined using E. coli grown in TGY medium; Extract-based peptide rich medium containing about 200 nM Mn. At 3 kGy, supplementing 1 μM Mn ²⁺ with TGY did not increase E. coli resistance; supplementing 13 mM phosphate with TGY increased E. coli resistance by 800-fold; and 3 mM uridine or 384 mM (3 %) Supplementing DMSO to TGY increased the resistance of E. coli by a factor of 50. When these agents were combined at independently applied concentrations, the viability of E. coli exposed to 3 kGy increased 10,000-fold.

Example 4-Reconstituted Mn2 ⁺ peptide complex:
The extremely radioprotective Mn ²⁺ -decapeptide-phosphate complex is a consensus amino acid sequence (H-Asp-Glu-His-Gly-Thr-Ala-) of hundreds of peptides purified from Deinococcus radiodurans. Val-Met-Leu-Lys-OH) (SEQ ID NO: 1). The composition of the mixture that spontaneously forms the Mn ²⁺ complex is 3 mM (H-Asp-Glu-His-Gly-Thr-Ala-Val-Met-Leu-Lys-OH) (SEQ ID NO: 1), 1 mM MnCl _2. Contains 25 mM orthophosphate (Pi) buffer (pH 7.4). When reconstituted in vitro, the Mn ²⁺ complex preserved the activity of the enzyme exposed to 50,000 Gy. Testing with decapeptide is important for its radioprotective properties when it is an amino acid composition of decapeptide and not a special sequence of amino acids, when combined with Mn ²⁺ and orthophosphate buffer. It proved that there is. The peptide need not be limited to 10 amino acids, but instead must be composed of specific amino acids present in the decapeptide.
Example 5-Application of reconstituted Deinococcus radiodurans Mn2 ⁺ complex for the production of irradiated vaccines

Bacterial irradiation using the method described herein was tested and demonstrated at 40,000 Gy using a model bacteriophage λ virus (FIG. 5). Mn ²⁺ complex (Mn-pep-Pi): 3 mM (H-Asp-Glu-His-Gly-Thr-Ala-Val-Met-Leu-Lys-OH) (SEQ ID NO: 1), 1 mM MnCl ₂ , 25 mM ortholine DNA was prepared from irradiated bacteriophage λ treated or not with acid salt (Pi) buffer (pH 7.4). At the indicated gamma dose (0-40 kGy), DNA (48.5 kbp genome) was purified from bacteriophage λ, subjected to conventional agarose gel electrophoresis, and then subjected to Southern blotting with a radiolabeled λ DNA probe. . As shown in FIG. 5A, the Mn ²⁺ complex does not significantly protect the DNA packaged in the virus.

The same bacteriophage λ preparation as examined in FIG. 5A was tested for protein integrity by separating the viral proteins using polyacrylamide gel electrophoresis. As shown in FIG. 5B, proteins in virus irradiated in the absence of Mn ²⁺ complexes were progressively destroyed. In contrast, proteins in virus samples containing Mn ²⁺ complexes were not affected by doses as high as 40 kGy.

At 40,000 Gy, the dose that caused viral DNA to disappear (see FIG. 5A), making the virus completely non-infectious, the viral protein was still sufficiently immunogenic. This was tested by Western analysis, which caused the λ protein to be challenged with antibodies produced in rabbits against non-irradiated λ phage. Identical positive results for immunogenicity were obtained for equivalent Westerns probed with antibodies raised against λ phage exposed to 40,000 Gy in the presence of Mn ²⁺ complexes. In contrast, λ phage exposed to 40,000 Gy in the absence of Mn ²⁺ complex did not raise antibodies in rabbits with significant specificity for native bacteriophage λ.

The approach was also successfully tested for pathogenic S. aureus strains (FIG. 6). In contrast, viruses and bacteria exposed to superlethal doses of IR without the Mn ²⁺ complex resulted in substantial loss of viral epitope integrity and loss of immunogenicity.

  Although the invention has been described and illustrated by reference to various specific materials, techniques and examples, the invention is not limited to the specific combinations of materials and techniques selected for that purpose. It is understood that it is not. Numerous changes in such details may be implied as will be appreciated by those skilled in the art. It is intended that the specification and examples be considered as exemplary only, with a true scope and spirit of the invention being indicated by the following claims. All references, patents and patent applications mentioned in this application are hereby incorporated by reference.

Claims (24)

A method for producing a vaccine against microorganisms comprising:
a) culturing, recovering and / or suspending microorganisms in the presence of a radioprotective composition, wherein said composition comprises an antioxidant and at least one peptide having 25 or fewer amino acids The peptide comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 1;
b) A method comprising irradiating the microorganism with a dose of radiation sufficient to render the microorganism incapable of replication.   The method of claim 1, wherein the radiation is selected from the group consisting of ultraviolet radiation, alpha radiation, beta radiation, gamma radiation, X-rays and neutron radiation.   The method according to claim 1 or 2, wherein the composition further comprises at least one nucleoside selected from the group consisting of adenosine, uridine, β-pseudouridine, inosine, and mixtures thereof.   4. The method of claim 3, wherein the at least one nucleoside is adenosine and uridine.   The method of claim 4, wherein the concentration of the at least one nucleoside is from about 1 mM to about 15 mM.   The method according to claim 1, wherein the at least one antioxidant comprises manganese.   The method of any of claims 1-6, wherein the concentration of the at least one antioxidant is from about 1 mM to about 12.5 mM manganese. Wherein the at least one antioxidant A method according to claim 7, wherein the member selected from the group consisting MnCl _2, and manganese phosphate.   The method according to any one of claims 1 to 8, wherein the composition further comprises at least one amino acid selected from the group consisting of alanine, valine and leucine.   The method according to claim 1, wherein the composition further comprises a phosphate.   11. A method according to any one of the preceding claims, wherein the composition further comprises an ultrafiltrate from D. radioodurans.   12. A method according to any preceding claim, wherein the radiation dose is at least about 20 kGy.   The method according to claim 1, wherein the microorganism is a bacterium.   The method according to claim 1, wherein the microorganism is a virus.   A method for making bacteria in culture resistant to ionizing radiation (IR), wherein the bacteria are cultured in the presence of a radiation protective composition, wherein the composition comprises at least one nucleoside, the number of which is also 1 One antioxidant, phosphate, dimethyl sulfoxide (DMSO) and at least one peptide having 25 or fewer amino acids, said peptide comprising an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 1. Inclusion method.   The method of claim 15, wherein the at least one nucleoside is selected from the group consisting of adenosine, uridine, β-pseudouridine, inosine, and mixtures thereof.   The method according to any one of claims 15 to 16, wherein the at least one nucleoside is adenosine and uridine.   18. The method of any of claims 15-17, wherein the concentration of the at least one nucleoside is from about 1 mM to about 15 mM.   The method according to any of claims 15 to 18, wherein the at least one antioxidant comprises manganese.   20. The method of any of claims 15-19, wherein the concentration of the at least one antioxidant is from about 1 mM to about 12.5 mM manganese. Wherein the at least one antioxidant The method of claim 20, wherein is selected from the group consisting MnCl _2, and manganese phosphate.   The method according to any one of claims 15 to 21, wherein the composition further comprises at least one amino acid selected from the group consisting of alanine, valine and leucine.   23. The method of any of claims 16-22, wherein the composition further comprises an ultrafiltrate from D. radioodurans.   The method according to any one of claims 16 to 23, wherein the bacterium is Escherichia coli.